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Introduction: Fc receptor-homolog 5 (FcRH5) is an immunoglobulin (Ig) domain-containing type I membrane protein that is expressed exclusively in the B-cell lineage. FcRH5 expression is retained in myeloma cells, with near 100% prevalence, and is elevated vs normal B cells. BFCR4350A is a humanized IgG-based T-cell-engaging bispecific antibody. Binding of BFCR4350A to the most membrane-proximal domain of FcRH5 on myeloma cells and to cluster of differentiation 3 (CD3) on T cells results in efficient immunological synapse formation and potent T-cell-directed killing of myeloma cells (Li, et al. Cancer Cell 2017). An ongoing Phase I dose-escalation study (GO39775; NCT03275103) is investigating the safety, activity, pharmacodynamics (PD) and pharmacokinetics of BFCR4350A monotherapy in patients (pts) with relapsed/refractory (R/R) multiple myeloma (MM). In Arm A, clinical activity was observed at the 3.6mg/20mg (step/target) dose level and above, and toxicity was manageable (Cohen, et al. ASH 2020). We present preliminary biomarker data that demonstrate the mode of action (MOA) of BFCR4350A, provide support for Cycle (C) 1 step-up dosing, and offer preliminary insights into markers that may predict response. Methods: In Arm A, R/R MM pts receive BFCR4350A by intravenous infusion in 21-day cycles. In C1, a single step-up dosing approach is used to mitigate the risk for cytokine release syndrome (CRS), with the step dose given on C1 Day (D) 1 and the target dose given on C1D8. The target dose is then administered on D1 of each subsequent cycle. PD changes in peripheral blood (PB) are assessed at baseline and at multiple time points within C1 by whole blood flow cytometry, plasma cytokine electrochemiluminescence and digital ELISA. Tumor biomarkers are assessed at baseline and pre-C2 by bone marrow (BM) biopsy dual CD138/CD8 immunohistochemistry staining and BM aspirate flow cytometry. The clinical cut-off date used for the current analyses was April 13, 2020. Results: At cut-off, all pts in Arm A (n=51) were biomarker evaluable. FcRH5 expression on myeloma cells was detected in all pts. Dose-dependent PD changes in PB were observed 24-192 hrs after the C1D1 infusion. Variable reduction in circulating T cells was observed 24 hrs after the 0.3-1.8mg C1D1 doses, while consistent reduction was observed after the 3.6mg C1D1 dose, with recovery by C1D8 (192 hrs). T-cell activation was detected 24 hrs post-infusion by upregulation of CD69 in CD8 and CD4 T cells and elevation of IFN-γ in plasma (median increase of ~150-fold from baseline), while T-cell proliferation (Ki67+) peaked by C1D8. At the 3.6mg C1D1 dose, CD8 T-cell activation and proliferation were up to 20-fold higher than at baseline. Minimal elevation of IL-6 was observed post-infusion in pts who received doses below 3.6mg on C1D1, while more consistent increases (≥100pg/ml) were observed in pts who received 3.6mg. IL-6 levels peaked within 24 hrs of the C1D1 dose and the kinetics of IL-6 increase were associated with dose and risk for CRS. Step-up dosing mitigated the risk for severe CRS, as evidenced by lower IL-6 levels after the C1D8 target dose compared to the 3.6mg C1D1 step dose in 27/33 (82%) pts (see Cohen, et al. ASH 2020 for corresponding clinical data). Preliminary data suggest that pts who respond to BFCR4350A have more pronounced T-cell expansion in PB, irrespective of baseline CD8 T-cell levels during the first week of C1. Analysis of the subset of pts with paired BM biopsies (n=19 pts) revealed that levels of CD8+ tumor infiltrating T-cells (TILs) were higher in responding pts than in non-responding pts at the end of C1. Conclusions: In this study, we demonstrated that early PD changes in T-cell activation, proliferation, and cytokine production confirm the MOA of BFCR4350A and support C1 step-up dosing for CRS mitigation in R/R MM. Early data suggest that at the end of C1, higher peripheral CD8 T-cell expansion and TILs are observed in responding pts than in non-responding pts. Additional analyses are ongoing to establish BFCR4350A response predictors and to better characterize the populations that are likely to benefit. Updated data will be presented. Disclosures Nakamura: Genentech, Inc.: Current Employment; F. Hoffmann-La Roche: Current equity holder in publicly-traded company. Lear:F. Hoffmann-La Roche: Current equity holder in publicly-traded company; Genentech, Inc.: Current Employment. Wilson:Genentech, Inc.: Current Employment. Koeppen:Genentech, Inc./ F. Hoffmann-La Roche: Current Employment; F. Hoffmann-La Roche, Pliant Therapeutics, Allogene, Jounce: Current equity holder in publicly-traded company. Vaze:Genentech, Inc./ F. Hoffmann-La Roche: Current Employment, Current equity holder in publicly-traded company. Trudel:Takeda: Honoraria; Sanofi: Honoraria; GSK: Consultancy, Honoraria, Research Funding; Genentech, Inc.: Research Funding; BMS: Consultancy, Honoraria, Research Funding; Karyopharm: Honoraria; AstraZeneca: Honoraria; Pfizer: Honoraria, Research Funding; Amgen: Consultancy, Research Funding; Janssen: Honoraria, Research Funding. Spencer:Amgen, Celgene, Haemalogix, Janssen, Servier and Takeda: Research Funding; AbbVie, Amgen, Celgene, Haemalogix, Janssen, Sanofi, SecuraBio, Specialised Therapeutics Australia, Servier and Takeda: Honoraria; AbbVie, Celgene, Haemalogix, Janssen, Sanofi, SecuraBio, Specialised Therapeutics Australia, Servier and Takeda: Consultancy; Celgene, Janssen and Takeda: Speakers Bureau. Harrison:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Patents & Royalties: wrt panobinostat; Janssen: Honoraria; BMS: Consultancy, Honoraria; CRISPR Therapeutics: Consultancy, Honoraria; Haemalogix: Consultancy; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Cohen:Novartis: Other: Patents/Intellectual property licensed, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda,: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees. Fine:Genentech, Inc.: Current Employment; F. Hoffmann-La Roche: Current equity holder in publicly-traded company. Li:Genentech, Inc./ F. Hoffmann-La Roche: Current Employment. Cooper:Genentech, Inc./ F. Hoffmann-La Roche: Current Employment, Current equity holder in publicly-traded company. Sumiyoshi:Genentech, Inc.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. OffLabel Disclosure: BFCR4350A is a humanized IgG-based T-cell-engaging bispecific antibody that targets the most membrane-proximal domain of FcRH5 on myeloma cells and CD3 on T cells. Dual binding facilitates efficient immunological synapse formation, resulting in T-cell activation and killing of myeloma cells. BFCR4350A is an investigational agent.

Introduction:TIGIT (T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif [ITIM] domain) is an inhibitory immunoreceptor expressed by T and natural killer (NK) cells that is an important regulator of anti-tumor and anti-viral immunity. TIGIT shares its high-affinity ligand PVR (CD155) with the activating receptor CD226 (DNAM-1). We have recently shown that TIGIT blockade, together with PD-L1/PD-1 blockade, provides robust efficacy in syngeneic tumor and chronic viral infection models. Importantly, CD226 blockade abrogates the benefit of TIGIT blockade, suggesting additional benefit of TIGIT blockade through elaboration of CD226-mediated anti-tumor immunity, analogous to CTLA-4/CD28 regulation of T-cell immunity. Whether TIGIT and CD226 are expressed in patients with multiple myeloma (MM) and how TIGIT expression relates to PD-L1/PD-1 expression is unknown. Here we evaluate expression of TIGIT, CD226, PD-1 and PD-L1 in patients with MM to inform novel immunotherapy combinations. Methods:We performed multi-color flow cytometry (n = 25 patients), and multiplex qRT-PCR (n = 7) on bone marrow specimens from patients with MM to assess expression of TIGIT, CD226, PD-1, and PD-L1 on tumor and immune cells. Cells were stained with fluorescently conjugated monoclonal antibodies to label T cells (CD3, CD4, CD8), NK cells (CD56, CD3), plasma cells (CD38, CD45, CD319, CD56), inhibitory/activating receptors (PD-1, TIGIT, PD-L1, CD226), and an amine-reactive viability dye (7-AAD). Stained and fixed cells were analyzed by flow cytometry using BD FACSCanto™ and BD LSRFortessa™. Results:TIGIT, CD226 and PD-L1/PD-1 were detectable by flow cytometry in all patients with MM who were tested, with some overlapping and distinct expression patterns. TIGIT was commonly expressed by marrow-infiltrating CD8+ T cells (median, 65% of cells), CD4+ T cells (median, 12%) and NK cells. In contrast, CD226 was more commonly expressed by marrow-infiltrating CD4+ T cells (median, 74%) compared with CD8+ T cells (median, 38%). PD-1 was expressed by marrow-infiltrating CD8+ T cells (median 38%) and CD4+ T cells (median, 16%). TIGIT was co-expressed with PD-1 on CD8+ T cells (67%-97% TIGIT+ among PD-1+), although many PD-1-negative CD8+ T cells also expressed TIGIT (39%-78% of PD-1-negative). PD-L1 was also expressed by CD8+ (median, 23%) and CD4+ (median, 8%) T cells in addition to MM plasma cells (median, 95%), albeit with significantly lower intensity on T cells compared with plasma cells. The expression of TIGIT and PD-L1 mRNA was highly correlated (R2 = 0.80). Analysis of PVR expression will also be presented. Conclusions: TIGIT, CD226, PD-1, and PD-L1 were commonly expressed in MM bone marrow, but with different patterns. Among CD8+ T cells, the frequency of TIGIT+ T cells was almost twice that of PD-1+ T cells, whereas the majority of CD4+ T cells expressed CD226. TIGIT blockade may complement anti-PD-L1/PD-1 immunotherapy by activating distinct T-cell/NK-cell subsets with synergistic clinical benefit. These results provide new insight into the immune microenvironment of MM and rationale for targeting both the PD-L1/PD-1 interaction and TIGIT in MM. Disclosures Yadav: Genentech, Inc.: Employment. Green:Genentech, Inc.: Employment. Ma:Genentech, Inc.: Employment. Robert:Genentech, Inc.: Employment. Glibicky:Makro Technologies Inc.: Employment; Genentech, Inc.: Consultancy. Nakamura:Genentech, Inc.: Employment. Sumiyoshi:Genentech, Inc.: Employment. Meng:Genentech, Inc.: Employment, Equity Ownership. Chu:Genentech Inc.: Employment. Wu:Genentech: Employment. Byon:Genentech, Inc.: Employment. Woodard:Genentech, Inc.: Employment. Adamkewicz:Genentech, Inc.: Employment. Grogan:Genentech, Inc.: Employment. Venstrom:Roche-Genentech: Employment.

Bispecific antibodies that retarget cytotoxic T cell activity to kill cancer cells are currently under clinical evaluation. However, the molecular mechanism for how CD3-bispecific antibodies 'trigger' intracellular T cell signaling is not known. We demonstrate that bispecific antibodies invoke an equivalent biophysical mechanism of TCR triggering as that observed for the TCR/pMHC interaction, including target clustering and exclusion of CD45 phosphatase from the synapse. The dimensions of the target molecule play a key role in the efficiency of the synapse formation. However, we demonstrate that rational epitope selection can overcome the spatial inhibition caused by target molecules with a large extracellular domain and result in efficient synapse formation and highly potent T cell triggering. With this insight, we developed a novel T-cell dependent bispecific (TDB) antibody, anti-FcRH5/CD3 TDB, targeting the B cell lineage marker FcRH5 for multiple myeloma. Anti-FcRH5/CD3 TDB demonstrated cytotoxicity against human plasma cells and patient derived myeloma tumor cells at picomolar doses. Very low target expression level is sufficient to induce anti-FcRH5/CD3 TDB mediated killing, indicating broad activity in multiple myeloma where the prevalence of FcRH5 expression is 100%. In primates, anti-FcRH5/CD3 treatment resulted in complete depletion of tissue B cells and bone marrow plasma cells. Anti-FcRH5/CD3 TDB induces immunosuppressive feedback signaling, including PD1 up-regulation, which can be overcome by PD-L1 antibodies. These data demonstrate the potential for the anti-FcRH5/CD3 TDB, alone or in combination with inhibition of PD1/PDL1 signaling in the treatment of multiple myeloma and other B-cell malignancies. Disclosures Li: Genentech: Employment. Stagg:Genentech: Employment. Johnston:Genentech: Employment. DiCara:Genentech: Employment. Clark:Genentech: Employment. Cook:Genentech: Employment. Slaga:Genentech: Employment. Nakamura:Genentech: Employment. Luke:Genentech: Employment. Sukumaran:Genentech: Employment. Luis:Genentech: Employment. Ye:Genentech: Employment. Sumiyoshi:Genentech: Employment. Danilenko:Genentech: Employment. Lee:Genentech: Employment. Totpal:Genentech: Employment. Ellerman:Genentech: Employment. Hötzel:Genentech: Employment. Junttila:Genentech: Employment.